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Non-glycosidic and non-peptidic inhibitors of selectin, and the use thereof

a non-glycosidic and non-peptidic technology, applied in the direction of biocide, peptide/protein ingredients, drug compositions, etc., can solve the problems of complex synthesis pathway, difficult structural elucidation, and high cost of starting compounds

Inactive Publication Date: 2009-02-03
JOHANNES GUTENBERG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]The largest group by number of the sLeX-analogs as tested consists of monosaccharide-compounds, wherein, while maintaining the fucose, the other essential binding epitopes in the molecule were replaced by non-glycosidic structures. Most successful are molecules, wherein specific peptides at the fucose simulate the contribution to the binding of the galactose and the negative sialic acid. Some substances exhibit a similar, in individual cases even a markedly enhanced binding affinity to the selectins, compared with the sLeX (Wong, C. H., et al., Small molecules as structural and functional mimics of sLex tetrasaccharide in selectin inhibition: A remarkable enhancement of inhibition by additional negative charge and for hydrophobic groups. J. Am. Chem. Soc. 119 (1997) 8152-8158.). Starting from sLeX, additional binding epitopes were searched for. It was found through the derivatization of the sLeX, and the computer simulation of the binding at the lectin domain, that a hydrophobic substitution at the glycosidic hydroxy group or at the amine of the glucosamine drastically improved the binding properties. New derivatives with aromatic ring systems at the amine or branched chain fatty acids at the glycosidic residue of the glucosamines, but also simplified sLeX-analogs with fatty acid substitutions were successfully tested (Ramphal, J. Y., et al., Ligand interactions with E-Selectin. Identification of a new site for recognition of N-Acyl Aromatic Glucosamine substituents of Sialyl Lewis x. J. Med. Chem. 39 (1996) 1357-1360.), and partially exhibited a more than 10-fold stronger binding, when compared with sLeX.
[0014]It is therefore an object of the present invention to provide inhibitors of selectin which overcome the disadvantages of inhibitors of selectin that are known in the state of the art, and which, in addition are more stable and synthetically more accessible.
[0068]Furthermore preferred is the use for the treatment of inflammatory diseases. The inflammatory diseases that can be treated according to the invention comprise peritonitis and rheumatoid arthritis. Furthermore, the treatment of tumorous diseases is possible.

Problems solved by technology

The disadvantages of glycosidic inhibitors are the complex synthesis pathway, the high costs of the starting compounds, the difficult structural elucidation, the hydrolytic instability, and the relatively low binding affinity.
The simultaneous substitution of glucose and galactose gave rigid and stabile fucose-sialic acid-derivatives, which, due to the lack of the galactose-6-hydroxy group, nevertheless, gave no improved effect.

Method used

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  • Non-glycosidic and non-peptidic inhibitors of selectin, and the use thereof
  • Non-glycosidic and non-peptidic inhibitors of selectin, and the use thereof
  • Non-glycosidic and non-peptidic inhibitors of selectin, and the use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Static Adhesion Assay

[0081]Following seeding in 24 well-plates, the examination of the adhesion was performed using (bovine aorta endothelial cells) BAEC monolayers of the cell passage 1-3 seven days. Before performing the assay, the completeness and the uniformity of the BAEC-monolayer was microscopically ensured. The culture medium (500 ml of medium M199, to which 6 ml of penicillin / streptomycin solution 1%, 6 ml L-glutamine (200 mM) and 100 ml (20%) of fetal calf serum, Sigma, Munich, were added) was removed, and the monolayer was washed twofold with DPBS (1000 μl / well, 37° C.). Subsequent to washing, the cells were treated with the cell culture medium containing 20 ng / ml of recombinant tumor necrosis-factor-alpha (TNFα) (in the presence of the respective test substance, dissolved in dimethylsulfoxide (DMSO) or only with DMSO as vehicle) for 20 minutes at 37° C. and 5% CO2 for 4 hours. After another twofold washing with DPBS (1000 μl / well, 37° C.) 900 μl of the culture medium and...

example 2

Visualization and Cell-Counting

[0084]The BAEC-sections were analyzed with an inverse Leica DM IRS-microscope (Leica, Bensheim, Germany) at 100-fold magnification. The analysis was performed in the centre of each monolayer, where the BAEC-layer with the adherent PMNs could be focused sharply. The pictures of the BAEC-sections were taken up with a resolution of 1200×1000 pixels using a Leica DC 200 digital camera that was connected to the microscope, and stored on a PC as bitmap (bmp) or as tagged image file format (tiff) by means of the Leica picture archive-software (Leica Mikrosysteme, Bensheim, Germany). For the manual counting of PMNs, several randomly selected sections of 0.25 mm2 in size from the centre of the respective well of the well-plate were counted. The counting took place using the picture archive-software IM 1000. (Leica Mikrosysteme, Bensheim, Germany). In order to do so, each PMN in the area as chosen was counted by a PC-mouse click, and the counted cells were label...

example 3

Analysis of Pictures

[0085]The analysis of the pictures was performed using the publicly available ImageJ-software (available at http: / / rsb.info.nih.gov / ij), installed on a standard-PC (Maxdata, Mergel, Germany). All pictures as obtained in a test run were grouped into an series, and then analyzed in an automated method using a specifically programmed macro. During the analysis, the colored picture was first transformed into an 8-bit picture. Subsequently, the allocation of the grey tones of each picture was plotted, the threshold of the grey tones was defined, and finally the cells were counted. In order to do so, the macro “of the customer-specific particle analysator” (authors: Roos, G., Rasband, W.; http: / / rsb.info.nih.gov / ij / plugins / particle-analyzer.html) was used. Counted PMNs were displayed as filled black circles. For all particles, the following parameters were recorded, area in pixels, minimum / maximum / average of the grey tones and the circularity (4π·(area / circumference2))...

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Abstract

A new class of non-glycosidic and non-peptidic inhibitors of selectins with low molecular weight according to the general formula 1is described, as well as methods for their production. These compounds represent a new class of non-toxic, in vivo anti-inflammatory effective inhibitors of selectins, and do not exhibit the disadvantages of the glycosidic inhibitor complexes, and are furthermore more potent in vitro, compared to the known drug bimosiamose. Furthermore, medicaments containing the compounds and their uses in the treatment of diseases are described.

Description

CROSS-REFERENCE TO A RELATED APPLICATION[0001]This application is a National Stage Application of International Application Number PCT / EP2005 / 008125, filed Jul. 26, 2005; which claims priority to German Application No. 10 2004 036 213.0, filed Jul. 26, 2004.DESCRIPTION[0002]The present invention relates to a new class of non-glycosidic and non-peptidic inhibitors of selectin with low molecular weight. The invention furthermore relates to a method for producing said compounds, medicaments containing these, and their use in the treatment of diseases.BACKGROUND OF THE INVENTION[0003]Selectins are carbohydrate-binding adhesion molecules that, during the process of an immune defense, contribute to an increased adhesion of leukocytes to the vessel endothelium of the inflamed tissue. They are divided into L-(leukocytes), E-(endothelium) and P-(platelets and endothelium) selectin, in accordance with their cells of origin. They initiate the adhesion of leukocytes through their protein struct...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07C205/00C07C229/00C07C249/00
CPCC07C235/24C07C251/54A61P29/00A61P43/00
Inventor DANNHARDT, GERDULBRICH, HOLGERPRECH, PHILIPLUXENBURGER, ANDREAS
Owner JOHANNES GUTENBERG UNIV